Membrane proteins play vital roles in many facets of biology and, due to their association with a variety of diseases, are of great therapeutic interest(1). Mechanistic understanding of membrane protein function depends critically on knowledge of their physical interactions and organization into multi-protein complexes. Despite extensive research in the past decade, there is a lack of in-depth information about the various interacting partners of these integral membrane proteins, mainly due to their biochemical features, enormous complexity and multiplicity(2). This consequently represents a major obstacle in attempts to investigate the biology of deregulation of integral membrane proteins leading to numerous human diseases such as cancer, cystic fibrosis, cardiovascular and neurodegenerative disorders and therefore also become an obstacle in designing improved targeted therapies.
To tackle the above-mentioned problems regarding the proteomic analysis of integral membrane proteins, the inventors previously developed the Membrane Yeast Two-Hybrid (MYTH) system (EP 1348766), a yeast-based technology for identification of protein interactors of integral membrane proteins from an organism of interest(3). Since its development, MYTH has been successfully applied to study protein-protein interactions (PPIs) among various membrane proteins from yeast, plant, worm and humans(4-10).
Despite the fact that MYTH is a powerful and robust system suitable for mapping the interactions of a wide-range of membrane proteins, our extensive experience in membrane proteomics during the past 10 years has taught us that many mammalian integral membrane proteins cannot be analyzed using MYTH. A potentially significant limitation is the host organism, yeast, which is used for identifying membrane PPIs. Specifically, yeast does not carry out some of the post-translational modifications (e.g. most tyrosine-phosphorylation events as well as some glycosylation events) that are responsible for mediating PPIs between many integral membrane proteins. In addition, the composition of the yeast cellular milieu and the membrane in particular (e.g. ergosterol in place of cholesterol) is different from that in mammals, which can result in improper localization of mammalian integral membrane proteins in yeast(11). Furthermore, a number of mammalian integral membrane proteins have been discovered to be toxic when expressed in yeast (Stagljar, I., unpublished data). Lastly, although yeast is a popular model organism for the elucidation of PPIs, it's not ideal for drug discovery purposes, such as the identification of small molecule drugs that can disrupt a PPI of therapeutic significance(12). All of the above-mentioned facts limit our ability to detect PPIs between mammalian integral membrane proteins and their interacting partners via MYTH and prevent us of using MYTH as a drug discovery tool. Thus, successful analysis of PPIs involving mammalian integral membrane proteins requires the development of a new technology that can detect these interactions in their natural membrane environment. Such a genetic system designed in a mammalian host organism would alleviate many of the above-mentioned concerns and provide an attractive method for uncovering the biological roles of many mammalian integral membrane proteins, which cannot be studied in yeast.
Transfer of the original yeast MYTH system into mammalian cells is not trivial and it requires significant protein engineering and modification, as use of original MYTH reagents are not compatible for mammalian cells.
Accordingly, what is needed is a genetic system that can probe PPIs involving mammalian integral membrane proteins, can detect stimuli-mediated PPIs or molecules that can disrupt PPIs, can detect the phosphorylation status of proteins. These and other needs, which cannot be met by the systems of the prior art, are now met by the present invention.